...a variety of popular plasmids and backbones with high cloning potential. By making these plasmids available...spectrophotometer. Other methods of quantification may yield slightly different DNA concentrations. Can I directly transfect...

... the results, techniques and reagents with the highest-possible quality of research output, RESOLUTE expects...production is used for assays and to generate antibody/ high-affinity ligands. Data is embedded in an integrated...

...We provide high quality AAV preps from select plasmids in the repository. Browse our AAV collection of...Larsen 107787 AAV.TBG.PI.Cre.rBGe TBG none 8 Wilson Light-Inducible Recombinases ID Name Promoter Fluorophore...

...Addgene determines that our current queue is too high and we do not expect to meet the general turnaround...complete any additional customs documentation that might be required by your country for the import of biological...

...pre-assembled genetic circuits such as logic gates and higher level gene networks. Sensing and Signaling Browse...Efflux Pumps Voigt Lab Repressors , Terminators , Light Signaling , Sigma Factors , Resource Allocator and...

...their large packaging capacity (>8 kb), high titers, and high levels of transgene expression. Additionally...

...γ-retroviruses. γ-Retroviruses can package relatively high amounts of DNA (up to ∼8 kb), and infection results...pRXTN CMV/MSV pRXTN is a modified version of pRX-tight Puro encoding NAT instead of PAC Iglehart 20672 ...

...Method Full Target Site (5' to 3') Left half-site Right half-site CACNA1F OZ507 and OZ508 OPEN OPEN gCGTGACCTCgctgatAAGAAGAGg...engineering of customized zinc-finger nucleases for highly efficient gene modification. Maeder ML et al. Mol...

... gRNA vectors suitable for prime editing are highlighted in the table below. Use the search bar to find...

...consider when planning experiments). What resources might be useful to help improve this technology or its...are some current obstacles that researchers face right now (e.g., not enough protocols, cost, not enough...

... 87429 TJP1-mEGFP AICSDP-23 mEGFP Tight junction protein ZO-1 Tight junctions 91565 AAVS1-mEGFP AICSDP...

...and C terminus and induces mutation at rates much higher than the parental vectors. pC-GoldyTALEN directs...obligate heterodimers that require cloning of left and right TALEN monomer proteins into opposite vectors. 37276...

...both P-elements and attB site for high expression of UAS-driven transgenes Worm unc-54,...Gateway cloning vectors Adenoviral High transduction efficiency, but do NOT integrate into...

...Parkinson's Richard Youle 23966 pTreTight-Htt94Q-CFP HTT CFP TREtight Huntington's Nico Dantuma 23971 ...Alzheimer's Gavin Wright 51643 APP-bio-His APP His, CD4 CMV Alzheimer's Gavin Wright 51811 PRNP-bio-His...PRNP-bio-His PRNP His, CD4 CMV Dementia Gavin Wright 53363 pCB268 APP Flag, HA, Myc CMV Alzheimer's Alexander Varshavsky...132389 AAVS1 CAG rtTA3 TauWT 2N4R-EGFP MAPT GFP TREtight Parkinson's, FTD Gerold Schmitt-Ulms 132393 AAVS1...AAVS1 CAG rtTA3 TauP301L 2N4R-EGFP MAPT GFP TREtight Parkinson's, FTD Gerold Schmitt-Ulms 133320 pHBS834...

...engineering using the CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Nat Protoc . ... engineered CRISPR-Cas9 complex. Konermann S*, Brigham MD*, Trevino AE, Joung J, Abudayyeh OO, Barcena...

...based on using a thin plane (sheet) of light generated by sunlight to observe single gold particles with...V= ventral Developments in light sheet fluorescence microscopy Modern light sheet fluorescence microscopy...out-of-focus reflected light by using a pinhole.  However, the excitation light excites all of the fluorophores..., there is a drop in image brightness with increasing depth caused by light scattering and absorption....of this century has seen some major advances in light microscopy, particularly related to the neurosciences....  One of these advances in microscopy has been light sheet fluorescence microscopy (LSFM). The underlying...a fluorescent sample and captured the reflected light using a different objective lens oriented perpendicular...

...fluorescence when exposed to 500 nm light.  Under 400 nm cyan light, Dronpa dimers dissociate, Dronpa goes...minute pulse of 500 nm light (turn on psRaf1) followed by 3 seconds of 400 nm light (turn off psRaf1). After...and use MEK1 as a model kinase. In this system, light is used to turn “on” and “off” MEK1 activity by ...is called psMEK1. When exposed to 500 nm violet light, the Dronpa dimers interact and lock MEK1 in an ... an inhibitor of choice, exposed to 500 nm cyan light to activate psMEK1, and the localization of ERK ...these same cells by 24-48 hours exposure to blue light also induced tail swelling in ~75% of worms (see...expressed in a tissue or organism that blue and violet light can penetrate. How can I try using photoswitchable...

...for many years (at a very low salary I might add). You have a right and responsibility to choose a lab where...Why is choosing the right lab such a big deal? It’s actually something you CAN choose and it will make...difference for your future career and life. You might see a lab head as choosing you, but in reality, ...communication before making this important decision. I’ll highlight some of these questions in the sections below....you are good at asking for and getting help, size might not be a big factor for you. Know who's boss You...considering a career at a small, teaching college you might want to consider less expensive, portable areas ...ownership" and author order PI lets lab members fight it out PI intervenes if there are interpersonal...

...experiments require higher gene expression and therefore higher titer than others. High titers are often ...vector to such a high titer when looking for effective gene expression. How do you achieve high titers in the...good ones into a cell to produce expression. The higher the titer, the better. AAV transport and transgene...transgene expression It’s clear that we need high titers, as contact between the cell and any individual AAV...the cell may not always be enough and therefore higher titer will be required. For example, retrograde...over an even larger area, thus necessitating even higher retrograde AAV titers for successful transduction...That means that once you drop below a certain (very high) threshold of viral particles per cell, you may ...

...the pressure to publish in high-impact journals, and the requirement for a high grant success rate. At this... this point, candidates are highly experienced but often have to start pursuing other options. While some...technical support specialist for 4 years. Read on for insights from my time in technical support including tips..., and resolve complaints about products. As you might expect, different companies produce different products... dialogue. This will give you a feeling of what might be involved in such a role. In addition, it can ...individual stories and get (sometimes refreshing) insights into how other scientists think and work. Most...to enhance the OD signals and then complain about high background. In many instances, variations of the...

...data on the assay methods. You might find that while the website highlights a variation of your application...protein or protein region highly conserved across species? If the proteins are highly homologous between your...

...antibodies have higher affinity and specificity than mouse monoclonal antibodies. Antibodies with higher affinities...monoclonal antibodies contain a single clone they have higher lot to lot specificity and tend towards more reproducible...When using a monoclonal antibody be sure to use a highly specific secondary antibody, rather than a broadly...at very low levels or your application requires a high degree of affinity, then species choice could very...may be the better choice for assays that require high affinity binding, such as immunoprecipitations. ...stain a target protein in mouse tissues, causes a high degree of background staining. This occurs because...antibody isotypes matter and helps you choose the right antibody for your application!  References and ...

...excitation light less efficiently. Similarly, decreasing the power of the excitation light reduces the...series described a practical approach to selecting a bright fluorescent protein. In the second post of this...destruction of a fluorophore under the influence of light. Any fluorescent molecule will photobleach at some...fluorescent proteins are excited with very intense light (for a brief time), whereas in widefield imaging...imaging fluorophores are excited with relatively low light levels (for a prolonged time). Other illumination...that, in experiments that measure photostability, high excitation powers are often used to reduce the length...linearly related to power, the conclusions reached at high power may not translate to real applications in ...

... short generation times and high fecundity, make zebrafish ideal for high throughput studies of optogenetics...Optogenetics, the use of light sensitive proteins (opsins) to manipulate cell activity, enables researchers... studies because it is non-invasive and offers a high degree of control over time and space. Zebrafish...imaging with fluorescent tags and activation of light sensing proteins. Furthermore, during the first ...Optogenetics functions through opsins which are light sensitive proteins. Opsins used in optogenetic studies...particles to cross the cell membrane when exposed to light. Neuronal firing depends on these charged particles...of the differing peak absorption wavelengths and light intensity requirements of different opsins, studies...

...solution for discriminating between neurons for light microscopy-based neuronal reconstruction. To achieve...et al., 2007; Cai et al., 2013). However, the brightness of Brainbow was inherently low. This is because...at ~2 copies per cell per color to achieve the highest color variations. Adjust the plasmid concentrations... to express a minimal amount of tTA to achieve highest expression levels. Clearing large tissues - Fluorescent...anatomical aspects of sensory circuit development using light microscopy. Lab website: https://dn-en.med.kyushu-u.ac.jp...clearing and index matching of tissue samples for High-resolution fluorescence imaging using SeeDB2." Bio-Protocol...Sakaguchi, Richi, Marcus N. Leiwe, and Takeshi Imai. "Bright multicolor labeling of neuronal circuits with fluorescent...

...separate proteins based on size. Whether higher molecular weight complexes are detected will depend on the...series described a practical approach to selecting a bright fluorescent protein and a photostable fluorescent... have a natural affinity and a tendency to form higher order aggregates. This property can be tolerated...purified fluorescent proteins to form homodimers or higher order oligomers can be analyzed by several techniques...essential to verify a number of key properties (brightness, photostability, oligomerization) of a number...will be using. If you do a head-to-head comparison right, it will be valuable information for the scientific...

...molecule. A higher brightness increases the detected signal and therefore high brightness is an important...EGFP). The higher the number, the higher the theoretical brightness. The theoretical brightness often appears...are (i) brightness, (ii) photostability and (iii) aggregation tendency. Brightness Brightness refers ...practical brightness is a better criterion than theoretical brightness for selecting the brightest fluorescent...and the sample. Practical brightness A protein with a high intrinsic brightness that does not fold well ...how the brightness is best defined or determined. Here, we will explain how practical brightness is determined...protein. Theoretical brightness Two key properties of fluorophores that determine brightness are the extent ...

...mutagenesis for the brightest teal fluorescent proteins that were photostable and had a high 480/530 nm emission...mWasabi, a bright, monomeric green fluorescent protein. In vitro, mWasabi is 1.6-fold brighter than EGFP...-type termini. mNeonGreen was reported as the brightest monomeric green or yellow fluorescent protein ...protein at the time. It is 1.5 to 3 times brighter than the most commonly used GFPs and YFPs. Its excitation...changes in pH. Find mTFP1 plasmids here! mWasabi: a bright green fluorescent protein The team that developed...Hui-wang, et al. "Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent...PMCID: PMC2292683. Shaner, Nathan C., et al. "A bright monomeric green fluorescent protein derived from...

...with the flick of a light switch. Neurons are not typically persuaded to fire when light is shined on them...expression of light-gated ion channels such as channelrhodopsins (ChRs) makes them light-responsive. When...mm3). Current channelrhodopsins also need high-intensity light for activation which requires implantation... no blue light, the channelrhodopsin remains inactive and neurons don't fire. With blue light, ions flow...are highly constrained and poorly understood. It’s hard to design new channelrhodopsins with high-throughput...requires higher expression levels in order to activate a neuron. ChRgers, however, have a higher conductance...the light was turned off.  Together these two experiments determined that ChRger2’s improved light sensitivity...

...dark state and highest fold induction at 31X. By using a slit pattern during blue light exposure (470nm...proteins N713-pMag and nMagHigh1-C714, this new system had both low background and high fold-induction of ...reversibly activated by turning blue light on and off. As one might expect from the modular nature of Cas9...fused with high-affinity interacting proteins Coh2 and DocS, respectively. The use of far-red light is of ...particular interest since blue light has limited applications in vivo. UV and blue light is readily absorbed and...tissues. Additionally, high intensity exposure can cause DNA damage. Far-red light, however, can penetrate...protein. Stop light! Photoactive anti-CRISPRs halt Cas9 activity Anti-CRISPR proteins are a highly diverse ...

...ever used Golden Gate Assembly for cloning, you might be familiar with the rules of thumb for designing...way to design GGA overhang sets, particularly for high-complexity reactions? The researchers over at New...otherwise! They suspected a data-driven approach might allow for an increase in the complexity. In 2018...need to be strictly followed in order to generate high-fidelity reactions. (Need a refresher? The five ...100% GC.) In fact, the NEB® team’s data showed that high-fidelity reactions could be done using overhang ...sets that broke rules 3—5. Fidelity, it turns out, might depend more on the individual sequence(s) than on...build out three tools to help researchers design high-fidelity Golden Gate Assemblies and overhang sets...

...Investigators, an open-access journal that enables high school students to publish peer-reviewed scientific...behind the journal and hear from a fantastic team of high school students who recently submitted to the journal...Journal of Emerging Investigators. Chapter 2 - Getting high school students and teachers interested in JEI (...mentorship programs. Chapter 6 and Outro - Interview with high school student researchers Arooba Ahmed, Jiachen...

...increasingly able to harness the body’s immune system to fight progressing cancers. It was inspiring to participate...from all fronts!”  Here I aim to compile a few highlights from the conference which reflect how cancer/...targeted engineering of the TCR locus. He also highlighted the UCSF Immunoprofiler - a human biopsy analysis...

...range between 0 and 100) are then converted to a weight (which ranges between 0 and 1) so that they can...ranging from -40 bp to 40 bp) are also converted to a weight (which ranges between 0 and 1) (shown in the graph...Conclusion CrispyCrunch is now publicly-available for high-throughput design and analysis of CRISPR experiments...

...researchers, we wondered how we could make the insights gained during this time more visible and thereby... ReFigure saves time, knowledge and makes your insights discoverable. Watch this quick video to learn ... ground again and again as they don’t save the insights they glean from a reading session. To remember...hunting through a whole folder of papers to find the right experiment! The ReFigure extension also tells readers...ReFigure making it easy for others to discover your insights on a topic. Beyond collating other’s data, you...include your own data in a ReFigure. For example, you might include a confirmatory image to show that a previously...previously used reagent performs well, or you might publish a replication experiment that you conducted ...

...hand, there are reasons why grad school might not be the right choice. I spoke with our Financial Operations...here. You also don’t have to go back to school right away. Shannon Rinaldi, Addgene’s Lab Manager, was.... I thought, ‘Let me pursue this, let me make a slight change so I can have flexibility and be up to date...biggest takeaways from my co-op. It has been so insightful to learn about different career paths, successes...

...oneself that prompted us to create Crowdfight.   Figure 1: Crowdfight is a community of scientists who...making science.   How we started Crowdfight-COVID19 Crowdfight was born as a collaborative platform...collaborator for a specific task, Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the expert...Ignacio Amigo and María Hernández Sánchez from Crowdfight. In March 2020, while most of the world was locked...did not have access to a P3 laboratory (BSL-3), a high-security facility needed to perform the experiments... them heard about our online platform, called Crowdfight, and filed a request for help. In just a few ... a research group in Scotland, who had just the right infrastructure and offered to collaborate with them...

...engineering of fusion proteins is relatively straightforward. However, generating a fusion while keeping...discuss strategies to generate fusion proteins and highlight some aspects of their design.  Protein size and...situation Next to size, potential homodimerization and higher order oligomerization of fluorescent proteins is... the fluorescent tag should operate as an inert light emitting module, any tendency of GFP to homodimerize...fluorescent protein characteristics such as the brightness, photostability, maturation time, and acid sensitivity...termini of your protein of interest The most straightforward fusion protein is made by connecting the N-...the fluorescent protein-POI fusions, we generate straight fusions, without any linker. When both orientations...

...and wrap the column with blue LED lights. When you switch off the lights, OptoNBs bind YFP and non-specific... the blue lights. Which option do you choose?  Option #2 may sound like fiction, but light-controlled...dark-induced dark-induced Loop 6 blue-light induced dark-induced blue-light induced Table 1: Summary of the... effect of AsLOV2 insertion site on light-induced binding.  Light controlled protein purification with...while cycling between dark and blue light which revealed a blue light-dependent shift in mCherry fluorescent...cells: light-based control of signaling pathways. This is particularly exciting because using light to control...binding of their target protein is altered by blue light illumination. This ability to reversibly bind and...

...the challenge of trying to figure out what system might work best for your particular purposes. We’re here...than others. In today’s post, we will focus on a high-level comparison of popular cloning methodologies...results. If you’re in a hurry, Golden Gate cloning might be for you. This methodology relies on versatile... in as little as 30 minutes! Alternatively, you might consider Gibson cloning. In this technique, you ...fragments in an order determined by their homology. You might also consider FastCloning, first described by Chaokun...approach should you use if the budget is particularly tight? Most cloning processes will require a PCR reaction... job done, provided that you’ve already got the right enzymes laying around the lab. If not, you’ll be...

...conducted in Professor Cepko's lab and to get some insight into her management and mentoring styles. If you'd...

... focused on three different levels of assembly: High efficiency and accurate single-insert assembly Intermediate...destination plasmid. Antibiotic selection allowed for high throughput cloning efficiency measurements. We performed...confirmed the insertion of the Lambda insert at the same high frequencies as the antibiotic selection markers....lacZ cassette assembly was easily achievable with high levels of transformants and low backgrounds – so...Using these 4 bp overhang observations, accurate “high-fidelity” junction sets for both the 12- and 24-...complex assemblies were performed. It was found that high efficiencies and accurate assembly levels were indeed...in the number of transformants while maintaining high levels of correct assemblies) translate to faster...

... (blue light-inducible AraC dimers in E. coli) that turns expression on and off with blue light.   Inspired...domain with VVD, a light-responsive domain that dimerizes upon exposure to blue light (Fig. 1). Another...VenturaIf you follow Barbara Di Ventura on Twitter, you might have seen the video of her singing about the paper...Another blue light-inducible dimerization domain, Vaucheria frigida aureochrome1 (VfAu1), led to functional...most applications.   Figure 1: With blue light, the VVD domains of the BLADE dimerize and activates...under the pBAD promoter, can be controlled with light without the need of any cloning.     What advantages...BLADE activates transcription with the flick of a light switch and shuts off just as quickly. The original...

...This is where FLEx enters the scene. FLEx: A light when night is about you! The FLEx (for flip-excision)...optogenetics? Optogenetics: Controlling neurons with light In 2005, a team from Stanford University, led by...ago with the discovery of microbial rhodopsins, light-sensitive proteins that absorb photons for energy...maintain membrane potential in response to visible light (Zhang et al., 2011). What do microbial rhodopsins...2000). Most neurons in the brain are not naturally light-sensitive, so the selective expression of opsin ...Optogenetic neuromodulation is made possible by the light: when hit with the correct wavelength, microbial..., 2011; Mei and Zhang, 2012). For example, blue light activates channelrhodopsins that, in turn, trigger...

...showed that improved specificity of Cas14 enables high-fidelity SNP genotyping. The development of  Cas14...

...Scientist. I used to spend my days (and often my nights :D) engineering E. coli to produce biofuels in ... screaming from the lab like the cartoon on the right?!? I would love to tell such a dramatic story, but...This may come as somewhat of surprise to many of my high school friends. In my teenage years I was super ...can involve walking alongside it and giving it a high five. While screaming can be therapeutic and I’ve...

...regulatory elements, pre-assembled genetic circuits, and higher level gene networks. Examples Include: promoters...

... NETRF believes that creating and maintaining a highly collaborative NET research community is pivotal...

...and Cpf1, which require that each DNA target have high sequence identity to the spacer sequence and contain...

...CRISPR-based purification methods are more straightforward and enable direct identification of molecules...